Detection of RNA expression from pseudogenes and non-coding genomic regions of Mycobacterium leprae.

We have previously reported that some pseudogenes are expressed in Mycobacterium leprae (M. leprae), the causative agent of leprosy, and that their expression levels alter upon infection of macrophages. We attempted to further examine the expression of pseudogene and non-coding genomic region in M. leprae, in this study. 19 Pseudogenes, 17 non-coding genomic regions, and 21 coding genes expression in M. leprae maintained in the footpads of the hypertensive nude rat (SHR/NCrj-rnu) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of some of these pseudogenes, non-coding genomic regions and coding genes were also examined in M. leprae from skin smear specimens obtained from patients with lepromatous leprosy by RT-PCR. Transcripts from pseudogenes, non-coding genomic regions and coding genes examined in this study were clearly observed in M. leprae. The expression patterns of some of these transcripts vary greatly among different leprosy patients. These results indicate that some of pseudogenes and non-coding genomic regions are transcribed in M. leprae and analysis of RNA expression patterns including pseudogene and non-coding genomic region in M. leprae may be useful in understanding the pathological states of infected patients.

[Comprehensive analysis of RNA expression of Mycobacterium leprae and clinical and biological significance].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. This result indicates that M. leprae is a very unique organism, and this future is important to understand the biological nature and/or pathogenicity of M. leprae, which remain unclear. We attempted to find the biological nature of M. leprae by detecting the gene and pseudogene regions transcribed at high level. We detected the genomic regions including pseudogenes and demonstrated that six out of twelve high expression regions were pseudogenes. In addition, its transcription level was changed when M. leprae infects macrophage. RNA was detected from genes, pseudogenes and non-coding regions. The expression levels of these regions were different among patients and a part of them is disappeared just after treatment. These results suggested that RNA derived from pseudogene and non-coding region have some function concerning the infection and/or intracellular parasitism and that the analysis of pseudogene and non-coding region expression pattern of M. leprae is available as a criterion for therapeutic effect and disease type of leprosy, and a prognostic marker.

High-level expression of pseudogenes in Mycobacterium leprae.

Recent studies have revealed that some RNAs are transcribed from noncoding DNA regions, including pseudogenes, and are functional as riboregulators. We have attempted to assess the gene expression profile throughout the Mycobacterium leprae genome using an array technique. Twelve highly expressed gene regions were identified that show an alteration in expression levels upon infection. Six of these were pseudogenes. Although M. leprae has an exceptional number and proportion of pseudogenes among species, our results suggest that some of the M. leprae pseudogenes are not just 'decayed' genes, but may have a functional role.

Congenital Erythropoietic Porphyria: Mutation of the Uroporphyrinogen III Cosynthase Gene in a Vietnamese Patient.

Congenital erythropoietic porphyria (CEP) arises from an autosomal recessive inherited disorder of the porphyrin metabolism, which leads to the accumulation of uroporphyrinogen I in bone marrow, skin and several other tissues by a deficiency of uroporphyrinogen III cosynthase (UROS). We studied a Vietnamese patient and her family suffering from severe cutaneous photosensitivity with skin fragility, bullous lesions and hypertrichosis on light-exposed areas. A missense mutation in the UROS gene was identified as a transversion of G to T at nucleotide 11,776, resulting in a substitution of valine by phenylalanine at codon 3 of exon 2. The patient showed a homozygous mutant profile, and the heterozygous state was observed in the parents. The activity of mutated UROS expressed in Escherichia coli was less than 16.1% that of the control, indicating that the markedly reduced activity of UROS is responsible for CEP. We described for the first time a mutation in the UROS gene in a Southeast Asian patient and a molecular diagnosis for the identification of clinically asymptomatic heterozygous mutation carriers and families with CEP.

Evaluation of polymerase chain reaction-based detection of Mycobacterium leprae for the diagnosis of leprosy.

Because Mycobacterium leprae cannot be cultivated in vitro, laboratory diagnosis of leprosy is generally made by microscopic and histopathological examination. The objective of the present study was to evaluate the sensitivity and utility of polymerase chain reaction (PCR) to detect M. leprae in comparison with other conventional methods for diagnosis such as split skin smears, histopathology and serodiagnosis. PCR amplification of the M. leprae-specific 16S ribosomal RNA was compared to other methods. Samples included 37 multibacillary (MB) patients with a positive bacteriological index (BI), 32 newly diagnosed paucibacillary (PB) patients whose BI were negative and 30 plaque psoriasis patients not residing in leprosy endemic areas as controls. The sensitivity of PCR was 30 fg of M. leprae DNA, which is equivalent to the DNA from 8.3 bacilli. The detection rate in MB and PB were 100% and 50%, respectively; the specificity was 100%. Semiquantitative evaluation of PCR correlated well with BI, but not with the morphological index (MI) nor with the serum antibody against phenolic glycolipid-1 (PGL-1). PCR detection of M. leprae targeting 16S ribosomal RNA was specific and more sensitive than conventional methods, and can contribute to early and accurate diagnosis of leprosy.